The objective of the proposed studies is to identify the cellular signals that regulate hepatocyte death in reponse to toxic liver injury. Our general hypothesis has been that cell death is not merely a passive process resulting from the biochemical effects of toxins but rather is actively regulated by cellular signaling cascades and changes in gene and protein expression. Critical to the hepatocyte's reponse to toxic injury are the death pathways activated by tumor necrosis factor-a (TNF) and reactive oxygen species (ROS). Studies completed over the last funding period have identified the c-Jun N-terminal kinase (JNK)/c- Jun/AP-1 signaling pathway as a central regulator of hepatocyte death from TNF and ROS. Preliminary data have demonstrated unique pro-apoptotic and pro-survival effects of different JNK isoforms in these two forms of hepatocyte death. Based on these studies and other preliminary data we propose the central hypothesis that hepatocyte survival or death in response to toxic injury is dictated by the integration of pro- and anti-apoptotic signals that are differentially regulated by distinct JNK isoforms. We will test this hypothesis by delineating the specific functions of different JNK isoforms in cultured hepatocyte and in vivo models of hepatotoxic injury in studies contained in four Specific Aims. First, we will test the hypothesis that JNK overactivation sensitizes hepatocytes to apoptosis from TNF by inducing E3 ligase-mediated degradation of C/EBPp. Second, we will test the hypothesis that toxins sensitize the liver to injury from TNF through JNK2-induced activation of the mitochondrial death pathway. Third, we will test the hypothesis that JNK1 and JNK2 have opposing effects on oxidant-induced hepatocyte death through their differential regulation of phospho-c-Jun. Fourth, we will test the hypothesis that the pro-apoptotic Bcl-2 family member Bim is the c-Jun-regulated downstream effector of hepatocyte death from oxidative stress. The ultimate goal of these studies is to further our understanding of the mechanisms of hepatocyte death and facilitate the development of therapies to prevent hepatic failure. Relevance to public health: The ultimate problem in every type of liver disease is the injury and death of hepatocytes that leads to the loss of hepatic function and liver failure. Understanding the mechanisms of hepatocyte death is critical to the prevention of hepatic failure from liver disease.